Cholecalciferol supplementation and angiogenic markers in chronic kidney disease

Vitamin D plays an important role in proliferation and differentiation of cells and deficiency of vitamin D disturbs angiogenic balance. Previous studies in animal models have reported an association between serum levels of vitamin D and balance between pro- and anti-angiogenic factors. There is insufficient evidence about the effect of vitamin D on mediators of angiogenesis in patients with CKD. We investigated the effect of cholecalciferol supplementation on serum levels of angiogenic markers in non-diabetic patients with CKD stage 3–4. In this secondary analysis on stored samples of our previously published randomized, double-blind, placebo-controlled trial, stable patients of either sex, aged 18–70 years, with non-diabetic CKD stage 3–4 and vitamin D deficiency (serum 25-hydroxyvitamin D ≤20 ng/ml) were randomized to receive either two directly observed oral doses of cholecalciferol (300,000 IU) or matching placebo at baseline and 8 weeks. The primary outcome was change in brachial artery flow-mediated dilatation at 16 weeks. Changes in levels of serum angiogenesis markers (angiopoietin-1, angiopoietin-2, VEGF-A, VEGEF-R, and Tie-2) between groups over 16 weeks were compared. A total 120 patients were enrolled. Supplementation with cholecalciferol led to significant improvement in FMD. Serum 25(OH)D levels were similar in both groups at baseline (13.21±4.78 ng/ml and 13.40±4.42 ng/ml; p = 0.888). At 16 weeks, the serum 25(OH)D levels increased in the cholecalciferol group but not in the placebo group (between-group difference in mean change:23.40 ng/ml; 95% CI, 19.76 to 27.06; p<0.001). Serum levels of angiogenic markers were similar at baseline. At 16 weeks, angiopoietin-2 level decreased in cholecalciferol group (mean difference:-0.73 ng/ml, 95%CI, -1.25 to -0.20, p = 0.002) but not in placebo group (mean difference -0.46 ng/ml, 95%CI, -1.09 to 0.17, p = 0.154), however there was no between-group difference at 16 weeks (between-group difference in mean change: -0.27 ng/ml, 95%CI, -1.09 to 0.55, p = 0.624). Serum angiopoietin-1 level increased [mean change: 5.63 (0.51 to 10.75), p = 0.018] and VEGF-R level decreased [mean change: -87.16 (-131.89 to -42.44), p<0.001] in placebo group but did not show any change in cholecalciferol group. Our data shows the changes in Ang-1, Ang-2 and Ang-1/Ang-2 ratio after high dose oral cholecalciferol supplementation in patients with non-diabetic G3-4 CKD. The data suggests changes in circulating levels of angiogenic markers which needs to be confirmed through an adequately powered study.

Introduction Angiogenesis, a physiological process that generally refers to the development of new blood vessels from pre-existing vessels [1], involves migration and proliferation of endothelial cells, lumen formation, maturation, and remodelling and is affected by a number of factors [2]. The balance between pro-angiogenic and anti-angiogenic molecules control the process of angiogenesis and imbalance of angiogenesis-related factors is implicated in the pathogenesis of various disorders like cancer, cardiovascular disease, kidney disease, and autoimmune diseases [3]. Many strands of evidence indicate that along with angiogenesis factors like angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), vascular endothelial growth factor-A (VEGF-A), vascular endothelial growth factor receptor (VEGFR), and Tie-2, vitamin D also plays a role in the regulation of angiogenesis [4]. Experimental studies have demonstrated an association between the balance of angiogenesis-related factors and the progression of CKD [5,6].
Vitamin D plays an important role in the proliferation and differentiation of cells by affecting the balance of angiogenesis-related factors. Previous studies have demonstrated that 1,25 (OH) 2 D 3 has antiangiogenic properties in vivo and in vitro, and inhibits angiogenesis in a tumor model [7]. Vitamin D supplementation studies showed protective effects on the incidence of preeclampsia by promoting angiogenesis in endothelial progenitor cells [8,9]. Vitamin D deficiency may disturb the angiogenic balance by increasing the production of angiotensin II, which further stimulates expression of Ang-2 and inhibits Ang-1 and Tie-2 leading to endothelial rarefaction in vitamin D deficient CKD [10,11]. Futrakul et.al. reported a reduction in levels of serum VEGF-A and Ang-1, and high Ang-2 levels in patients with CKD [12]. Another observational study demonstrated that the imbalance between Ang-1 and Ang-2 (high Ang-2 and low Ang-1) was associated with a subclinical cardiovascular abnormality in patients with CKD [13].
Not much is known about the effect of vitamin D on mediators of angiogenesis in vitamin D-deficient patients with CKD. In a prespecified secondary analysis of randomized, doubleblind, placebo-controlled trial of cholecalciferol supplementation in vitamin D-deficient, nondiabetic subjects with stage 3-4 CKD, we investigated the effect of oral cholecalciferol supplementation on serum levels of various angiogenic markers.

Materials and methods
The current study is a secondary analysis on stored biological samples of a parallel-arm, randomized, double-blind, placebo-controlled trial that investigated the effect of a high dose of oral cholecalciferol supplementation on vascular function [14]. The study was done at the Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India. Ethics approval was obtained from Institute Ethics Committee to use the stored biological samples of the trial for biomarker analysis. Prior provided written informed consent was taken from all study subjects. The authors confirm that all ongoing and related trials for this drug/intervention are registered. The detailed study protocol and subject enrolment process including the Consolidated Standards of Reporting Trials (CONSORT) flow diagram have been described earlier [14]. Briefly, 120 non-diabetic, CKD stage 3-4 subjects, age between 18 to 70 years and serum 25(OH)D levels �20 ng/ml were included in this study. A computer generated Bernoulli random number table was used for randomization of study subjects. Randomization was done in a 1:1 allocation ratio to receive two directly observed doses of either 300,000 IU of cholecalciferol or matching placebo at baseline and 8 weeks later. Follow up was done at 16 weeks after randomization. The time gap between cholecalciferol doses and analysis of variables is concordant with what has already been published [15].
Demographic, clinical, and biochemical parameters were recorded at baseline and 16 weeks. Flow-mediated dilatation was assessed at baseline and 16 weeks using a Philips IU22 xMatrix ultrasound system (Philips, Cambridge, MA) and automated continuous edge detection and wall tracking software (Brachial Analyzer for Research 6, Medical Imaging Applications LLC, Coralville, IA). Angiogenic biomarkers were analysed at baseline and 16 weeks in serum samples. Serum 25(OH)D and 1,25(OH) 2 D was measured by enzyme immunoassay [EIA; Immunodiagnostic Systems (IDS), UK], as described earlier [14]. Serum levels of Ang-1, Ang-2, vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR), and Tie-2 receptor were analysed using Quantikine 1 ELISA kit (R&D Systems, USA) as per manufactures protocol.

Outcomes
We compared changes in levels of angiogenesis biomarkers-Ang-1, Ang-2, VEGF, VEGFR, and Tie-2 receptor at 16 weeks from baseline after cholecalciferol supplementation.

Statistical analysis
Data in this study are presented as mean with standard deviation (SD), mean change (95% confidence interval) and frequency (percentage) as appropriate. Normally distributed continuous variables were compared with independent samples Student's t test, or Mann-Whitney U test. Categorical variables were analysed by Chi-Square or Fisher's Exact tests as appropriate. Paired Student's t-test and Wilcoxon signed-rank test were used for within-group comparisons for normally and non-normally distributed data, respectively. P-values <0.05 with two tailed were considered significant. Statistical Package for the Social Sciences (SPSS) software for Macintosh, version 21.0 (IBM Corp., Armonk, NY, USA) was used for all the statistical analysis.

Results
A total of 423 subjects were screened in this study and 120 were enrolled, randomized after exclusion of 303 subject due to various reasons (Fig 1). Out of 120 randomized patients, 59 and 58 patients in the placebo and cholecalciferol group respectively completed their follow up. The baseline demographic characteristics of the two groups are shown in Table 1. Participant demographics and causes of CKD were similar in both groups. Biochemical parameters and serum level of angiogenic markers were also similar in both groups at baseline ( Table 2).  Table 3). At 16 weeks, the FMD improved in the cholecalciferol group but not in the placebo group [14]. Table 3 Table 3). Serum Ang-1 levels  Table 3). The levels of serum VEGF and Tie-2 remained unchanged in cholecalciferol as well as placebo groups (Table 3). Further, we analysed the Ang-1/Ang-2 ratio but did not found any significant difference in mean change between groups (-3.32, 95% CI: -9.12 to 2.48, p = 0.563, Table 3).

Discussion
We have shown through this randomized double-blind placebo-controlled trial that oral highdose cholecalciferol supplementation can correct native vitamin D deficiency in stable nondiabetic, subjects with G3-4 CKD and induce a favorable change in FMD, a marker of endothelial function [14]. The main intention of the present study was to evaluate the effect of oral cholecalciferol supplementation on serum levels of angiogenic markers in subjects with CKD. Overall, we found no significant effects of a high dose of oral cholecalciferol supplementation on angiogenic markers in these subjects. Although the serum level of Ang-2 was reduced in the cholecalciferol group, the intergroup difference was not significant. There was no effect on the levels of other angiogenic markers such as Ang-1, VEGFR, VEGF, and Tie-2. Patients with CKD show impaired balance of angiogenic factors caused by an elevation in the circulating level of Ang-2, and reduction in level of Ang-1 [12]. Reduced Ang-1/Ang-2 ratio is a strong predictor of long-term mortality in CKD patients [16]. We found that cholecalciferol supplementation significantly reduced serum levels of Ang-2, as also shown in a recent study that reported a reduction in circulating levels of VEGF-A and Ang-2 in breast cancer patients after treatment with cholecalciferol supplementation [17]. We did not find any difference in the Ang-1/Ang-2 ratio between the two groups.
In vitro studies have also confirmed that treatment with vitamin D analogues decreased the expression of Ang-2 in cell lines [18,19]. In fact, some studies suggested that vitamin D supplementation could inhibit angiogenesis in tumor cells [7,20,21]. However, endothelial cells within the tumors are not the same as in normal tissues. In fact, vitamin D shows pro-  angiogenic effects in other disorders like preeclampsia, an early stage of CKD, and CVD [8,14]. Therefore, it is important to understand whether the antiangiogenic effect of vitamin D occurs in both tumor and normal tissues and whether these effects occur by direct or indirect actions on the signalling of angiogenesis. It is well known that Ang-1 is a key regulator of angiogenesis, and stabilizes endothelial cells, but adenoviral delivery of Ang-1 induced inflammatory and fibrotic responses in a mouse folic acid-induced nephrotoxicity model [22]. However, none of the studies has shown the effect of cholecalciferol supplementation on Ang-1 and Tie-2 except this current study and, results suggested that a high dose of oral cholecalciferol has no significance effect on these angiogenic markers.
The interaction between vitamin D and angiogenesis balance has been studied in other conditions. Oral vitamin D 3 supplementation reduces the risk for preeclampsia, a state of antiangiogenesis [23][24][25]. Irani et al. demonstrated a significant decrease in VEGF levels in vitamin D-deficient patients with polycystic ovary syndrome after vitamin D 3 supplementation [26]. There are some observational studies which reported the high circulating VEGF-A in CKD and diabetic nephropathy and a lower Ang-1/VEGF-A ratio is in patients with CKD [27,28]. VEGF levels were detected to be increased [29], decreased [30], or did not differ between patients with CKD and healthy controls [31]. But none of these studies has analysed the association or effect of vitamin D on VEGF levels. In our study, VEGF levels remained unchanged in both the groups suggesting that oral cholecalciferol supplementation did not significantly alter the expression of VEGF in patients with early CKD. Interestingly, VEGFR levels significantly declined in the placebo group, but remain unchanged in the cholecalciferol group. The significance of this finding is unclear.
The major strength of our study is the setting of a randomized, double-blind, placebo-controlled trial, homogenous study populations, and adequate sample size to estimate the primary outcome. The relatively short duration, analysis at only two time points, exclusion of subjects with diabetes and the post-hoc nature of the secondary analysis are an important limitation of this study. The study may not be significantly powered to detect differences in outcome parameters for this secondary analysis.
In conclusion, our data shows the changes in Ang-1, Ang-2 and Ang-1/Ang-2 ratio after high dose oral cholecalciferol supplementation in patients with non-diabetic G3-4 CKD. The data suggests changes in circulating levels of angiogenic markers which needs to be confirmed through an adequately powered study.